Process for preparing collagenous material tanned with polyacrolein



United States Patent Ofiice 3,093,440 Patented June 11, 1963 Thisinvention relates to the tanning of collagenous material of mammalianorigin in sheet or tubular form suitable for implanting permanently ortemporarily in a mammal, and more particularly relates to the tanning ofcollagenous material by the use of polyacrolein. A particularly suitablecollagenous material for use in the practice of this invention isdescribed in US. Patent No. 2,900,644, August 25, 1959, wherein it isdisclosed that such material may be obtained from mammalian tubularblood vessels and especially from a bovine tubular blood vessel fromwhich the objectionable parenchymatous protein is removed by digestionwith a proteolytic enzyme such as ficin.

In order for collagenous material and particularly tubular collagenousmaterial of mammalian origin to be suitable for implanting permanentlyor temporarily in a mammal, it must be tanned so that it has increasedresistance to breakdown by normal body processes and have a highresistance to rupture because of the normal flexes and strains to whichit is subjected during the time of its implantation. Short abdominalgrafts of tubular collagenous material prepared from bovine tubularblood vessels which have been tanned with tanning agents such as aqueousformaldehyde, glyoxal, and chromic oxide have been shown to have a highorder of success and resistance to rupture when implanted in dogs.However, when short segments of such tanned tubular collagenous materialof bovine arterial origin are used as thoracic and long segments areused as thoraco abdominal grafts in dogs, the incidence of rupturewithin one or two weeks following implantation has been found to beincreased significantly. This demonstrates that tanning of grafts withformaldehyde does not produce a bonding with sufficient durability toresist the natural breakdown processes of the body so that it isconsidered necessary to develop a tanning agent and tanning procedurefor tubular collagenous material of bovine arterial origin able toinsure a high degree of resistance to rupture when used as thoracic andthoraco abdominal grafts in dogs and particularly when used as longgrafts.

The discoveries associated with the invention and relating to thesolution of the above problems and the objectives achieved in accordancewith the invention as set forth herein include: The provision ofcollagenous material of mammalian origin in sheet or tube form tannedwith polyacrolein so that it has increased resistance to breakdown bynormal body processes; the provision of tubular collagenous material ofmammalian origin in Y or T form tanned by treatment with polyacrolein sothat it has increased resistance to breakdown by normal body processes;the provision of a process for preparing tubular collagenous material ofmammalian origin from which the objectionable parenchymatous protein issubstantially removed by digestion With a proteolytic enzyme to increasethe collagenous solid content to at least 80% and tanning to increaseresistance to rupture when used as an abdominal, thoracic or thoracoabdominal graft in dogs by treatment with polyacrolein; the provision ofa tubular collagenous material of mammalian origin substantially in itsnaturally-produced forrn containing at least 80% collagenous solids andnot over 20% objectionable protein and tanned to provide increasedresistance to rupture when used as an abdominal, thoracic or thoracoabdominal graft in dogs by treatment with polyacrolein; the provision ofsuch a process and followed by storing the washed product in a storagefluid such as 50% aqueous U.S.P. alcohol or isopropyl alcohol which maycontain a sterilizing agent such as 1% propylene oxide; and otherobjectives which will be apparent as details or embodiments of theinvention are set forth hereinafter.

In order to facilitate a clear understanding of the invention thefollowing specific embodiments are detailed.

Example 1 Twenty inch long segments of fresh beef carotid artery wereremoved between the origin at the innorninate and the cephalicbifurcation in the head immediately following carcass splittingoperation, washed in cold water, freed of adherent connective tissue,digested for two hours and fifteen minutes at 37 C. in a solution of onepercent (weight/volume) commercial ficin buttered at pH 5.1 with an 0.1M citric acid-sodium citrate buffer, washed for two hours in fivechanges of distilled water, treated for 2.4 hours with 0.1% aqueoussodium chlorite solution to inactivate any ficin which may have beenadsorbed to the collagen fibers of the digested artery, and immersed foreighteen hours at room temperature in a 0.5% (weight/volume), aqueous,polyacrolein tanning solution prepared by slowly bubbling gaseous sulfurdioxide with stirring into a suspension of two and one-half grams ofpolyacrolein, having an average molecular weight of 220,000, in ten cc.of water. The gaseous sulfur dioxide was bubbled into the supsensionuntil all of the polyacrolein was in solution and the solution had nogelling tendencies on standing. The pH of the resulting aqueous solutionof polyacrolein was adjusted to 6.0 with concentrated sodium hydroxideand the resulting solution was diluted with water to provide a 0.5%(weight/volume) tanning solution. The arteries after immersion in thetanning solution for eighteen hours were removed from the tanningsolution and washed with distilled water for two hours to remove excesstanning agent. The shrinkage temperature of tanned bovine arterysegments prepared as above came within the range of 83-88 C. Theshrinkage temperature of bovine artery segments prepared and tannedaccording to the above procedure is used as a measure of the degree oftanning obtained and also to determine the stability against reversal oftanning by sterilization procedures.

Shrinkage temperature (Ts), the temperature at which twenty percent ofthe potential shrinkage has occurred, is recommended in the tanningindustry to indicate the degree of tanning (Gustavson, K. H., TheChemistry and Reactivity of Collagen, Academic Press, N.Y., 1956, p.277) wherein it is stated that tanning of collagen increases itsshrinkage temperature and the shrinkage temperature is thereforeimportant in controlling tanning processes. The shrinkage temperature oftanned bovine artery segments was determined by the method and devicedescribed by McLaughlin and Theis (McLaughlin, G. D., and Theis, E. R.,Chemistry and Leather Manufacture, Am. Chem. Soc. Monograph No. 101, NewYork Reinhold Publishing Co., 1945, p. 135).

Bovine artery segments prepared and tanned according to the above methodwere sterilized by immersion for 14 days at F. in 50% aqueous ethanolcontaining 1% propylene oxide and then had a shrinkage temperaturewithin the range of 8388 C., which corresponds to the shrinkagetemperature before immersion in the sterilizing solution. The absence ofchange in shrinkage temperature range established that tanning withpolyacrolein results in the production of a tanned bovine artery segmentwhich is stable in the sterilizing solution.

A twenty-two pound mongrel dog was anesthetized with pentobarbitalsodium to achieve general anesthesia and a respirometer was attached tofacilitate breathing. Two incisions were made to permit insertion of athoraco abdominal graft. One was prepared in the left lateral aspect ofthe abdomen and the other was a left thoracotorny at the level of theseventh intercostal space. The left lung Was collapsed and respirationwas maintained in the right lung by intermittent positive pressure.

A graft prepared as described above and tanned for eighteen hours atroom temperature at pH 6.0 in 'a 0.5% (\t eight/volume) aqueouspolyac'rolei'n solution and sterilized as described had a shrinkagetemperature of 83 C. before sterilization and a shrinkage temperature of87 C. after sterilization. The sterile graft which had a length of 24.0centimeters and a diameter of 11.5 millimeters at both the proximal anddistal ends, was implanted in the anesthetized dog according to thefollowing procedure: An end to side anastomosis was first completed withthe graft and abdominal aorta at a point just above the inferiormesenteric artery. On completion of the anastomosis the graft wasthreaded through an artificial hiatus produced in the diaphragm. Thethoracic aorta was transected and the graft was anastomosed with theproximal cut end of the aorta. The distal end was closed with a doublelayer and remained as a blind pouch. The incisions were closed and thedog allowed to recover.

An aortogram was taken after three weeks, the dog was sacrificed and thegraft removed for gross and microscopic findings. The aortogram showedno change in diameter or shape of the graft. On gross inspection theadventitial coat appeared to show greater invasion of host tissue in theabdominal section than in the thoracic section. On microscopicinspection a minimal tissue reaction with evidence of fibroblastinvasion of the adventitia but not the media, was observed.

Example 2 A thirty pound mongrel dog was grafted with a thoracoabdominal graft as in Example 1. The graft, which had been prepared,tanned, and sterilized in the same manner as the graft used in Example1, was 20.7 centimeters long and had a diameter of 14.0 millimeters atthe proximal end and 13.0 millimeters at the distal end and had ashrinkage temperature before sterilization of 85 C. and a shrinkagetemperature after sterilization of 84 C.

An aortogram was taken at five weeks, the dog sacrificed, and the graftremoved for gross and rniscroscopic findings. The aortogram showed nodilatation. On gross examination the graft looked good with apparentadventitial invasion especially in the abdominal area. On microscopicexamination the graft appeared well infiltrated in all zones except forthe media. There were occasional lymphocytic foci on the periphery ofthe tissue surrounding the graft.

Example 3 A twenty-nine pound mongrel dog was grafted with a thoracoabdominal graft as in Example 1. The graft, which had been prepared,tanned and sterilized in the same manner as the graft used in Example 1,was 21.0 centimeters long and had a diameter of 13.5 millimeters at theproximal end and 13.0 millimeters at the distal end and had a shrinkagetemperature before sterilization of 85 C. and a shrinkage temperatureafter sterilization of 84 C.

An aortogram was taken in nine weeks, the dog sacrificed, and the graftremoved for gross and microscopic findings. The aortogram showed someirregularity of the intima in the thoracic end. On gross inspection itwas apparent that an aneurysmal dilatation had occurred at the flexedlower end of the graft. In addition there was a thick cheesy thrombus inthe lower end and a relatively thick one in the upper region. Ondissection the graft did not appear as good as the others although theadventitia was well infiltrated. There was some petechial hemorrhages atthe lower end. Microscopic examination showed poor invasion in someparts although adequate in others and a slightly undesirable tissueresponse characterized by the presence of lymphocytes and plasma cells.Portions of the mural thrombus were becoming organized and were verythick in some areas. In some zones the fibroblastic invasion was veryheavy but only minimal amounts of collagen had been deposited.

Example 4 A twenty-five pound mongrel dog was subjected to hypothermiato reduce body temperature to 31 C. The animal was anesthetized withpentobarbital sodium to achieve general anesthesia and a respirometcrwas attached to facilitate breathing. The thoracic aorta was approachedby a left thoracotorny at the level of the seventh intercostal space.The left lung was collapsed and respiration maintained in the right lungonly by intermittent positive pressure. The intercostals likely tointerfere with the procedure were tied and transected. A piece ofumbilical tape passed under the aorta and used as traction greatlyfacilitated identification and tying of the tributaries. Havingmobilized a desired length of aorta, non-crushing vascular clamps (Pottscoaraction forceps) were placed at the most proximal and distal freepoints and the section of artery between the clamps was resected. Carewas taken to leave sufficient cult to permit suturing. The two vascularclamps were placed in a dorsoventral manner. Their position tended toorient the graft at the time of anastomosis.

The proper length of graft (6.2 centimeters) required to fill the defectwas determined by accurate measurement and the prosthesis was cutaccordingly. The graft used was 6.2 centimeters long and had a diameterof 9.0 millimeters. The graft had been prepared, tanned and sterilizedin the same manner as the graft used in Example l and had a shrinkagetemperature before sterilization of 84 C. and a shrinkage temperatureafter sterilization of 87 C. Using 50 arterial silk swedgcd to a halfcircle needle, two guy sutures were placed through the lateral edges ofthe proximal cut end of the aorta and the graft. Mosquito forcepsattached to one end of the suture were allowed to hang outside theincision thereby creating traction. This helped to bring the line ofanastomosis into view and simplified the suturing procedure. The distalfree end of the graft was flipped dorsad to expose the underside of theanastomatic line. The lower half was closed with a continuous over andover stitch using one of the long needle ends of a previously placed guysuture. The graft was returned to the horizontal position and the upperhalf of the proximal anastomatic line was closed in a similar fashion.The graft was returned to the horizontal position and the upper half ofthe proximal anastomatic line was closed in a similar fashion. Thedistal anastomosis was started by placing two guy sutures in such amanner as to bisect the lumen laterally. Sutures so placed permittedrotation of the graft in a horizontal plane and allowed easy placementof sutures throughout the entire circumference. The anastomosis wasaccomplished in the same manner as the proximal end using a continuousover and over suture. On completion of the anastomosis, the distal clampwas released and the graft permitted to fill by retrograde flow. Theproximal clamp was then released to reestablish circulation. Hemorrhageoccurred at both suture lines but was arrested by wrapping the sutureline with a gauze sponge and gently applying pressure. The incision wasclosed and the dog allowed to recover.

After five weeks the dog was subjected to aortography to evaluate insitu appearance of the graft and then sacrificed. The graft was removedfor gross and microscopic findings. The aortogram showed no dilatation.On gross examination the graft appearance had not significantly changed.The suture line was covered with pseudo intima. On microscopicexamination it was determined that the adventiti'al coat was completelyinvaded with new fibrous tissue. A thick layer of collagen completelysurrounded the graft. Some of the original adventitial collagen bandswere visible. The intima was intact and smooth. A few fibroblasts hadinvaded the media but had not started to produce collagen.

Example 5 An eighteen pound mongrel dog was subjected to hypothermia toreduce the body temperature to 3 1 C. The animal was anesthetized withpentobarbital sodium to achieve general anesthesia and a respirometerwas attached to facilitate breathing. The graft inserted was 5.3centimeters long and had a diameter of 12.0 millimeters. A thoracicimplant operation was performed as in Example 4. The graft used in theoperation had been prepared, tanned and sterilized in the same manner asthe graft used in Example 1 and had a shrinkage temperature beforesterilization of 83 C. and a shrinkage temperature after sterilizationof 87 C.

After nine weeks the dog was subjected to aortography to evaluate insitu appearance, the dog was sacrificed and the graft was removed forgross and microscopic findings. The aortogram showed no dilatation. Ongross examination the graft did not appear to have been significantlychanged except there was noticeable adventitial invasion. On microscopicexamination it was observed that the surrounding collagen was about thesame thickness as at five weeks but was much more mature and it wasdifiicult to identify the original adventitial collagen. There were someplaques of acute inflammation not unlike a phlegmon. There were alsosome giant cells in this section and large macrophages with vesicularnuclei. These reactive areas were focal in distribution.

Example 6 Bovine arteries were prepared in the same manner as in Example1 except they are tanned by immersion for eighteen hours in an aqueous1.0% formalin solution buffered with 0.1 Molar sodium-potassiumphosphate at pH 7.2. The tanned grafts had shrinkage temperatures withinthe range of 86 C. to 91 C. before immersion in the sterilizing solutionand shrinkage temperatures within the range of 70 C. to 76 C. afterimmersion in the sterilizing solution. The substantial lowering of theshrinkage temperature following tanning by formalin showed that thetanned product was substantially less stable than the product tanned bypolyacrolein. The tanned grafts were sterilized in the same manner as inExample 1.

A twenty-eight pound mongrel dog was anesthetized with pentobarbitalsodium to achieve general anesthesia and maintenance of respiration wasachieved by means of a Burns type valve. The dog was grafted with athoraco abdominal graft prepared as above using the implantationtechnique described in Example 1. The graft used was 24.5 centimeterslong and had a proximal diameter of 11.0 millimeters and a distaldiameter of 10.0 millimeters and had a shrinkage temperature beforesterilization of 87 C. and a shrinkage temperature after sterilizationof 73 C.

The dog died eleven days postoperatively due to an ill-defined rupturein the thoracic cavity.

Example 7 A thirty-one pound mongrel dog was grafted with a thoracoabdominal graft using the implantation technique described in Example 1.The graft had been prepared, tanned and sterilized in the same manner asthe graft used in Example 6. The graft was 22.0 centimeters long and hada diameter of 11.0 millimeters at the proximal end and 10.0 millimetersat the distal end and had a shrinkage temperature before sterilizationof 86 C. and a shrinkage temperature after sterilization of 73 C.

The dog died seventeen days postoperatively due to a rupture in theabdominal cavity.

Example 8 A thirty pound mongrel dog was grafted with a thoracoabdominal graft using the implantation technique described in Example 1.The graft had been prepared, tanned and sterilized in the same manner asthe graft used in Example 6. The graft was 23.0 centimeters long and hada proximal diameter of 10.5 millimeters and a distal diameter of 10.5millimeters and had a shrinkage temperature before sterilization of 91C. and a shrinkage temperature after sterilization of 75 C.

After nine weeks the dog was subjected to aortography to evaluate insitu appearance of the graft, and then sacrificed for closer evaluationof its gross and miscroscopic appearance. The aortogram indicated nodilatation. Gross appearance indicated that the artery was in goodcondition and would be considered satisfactory at this stage. There wassome thinning at the proximal end possibly due to too severe dissectionat the time of autopsy. Pseudo intima extended from both suture linesfor 2 to 2.5 centimeters. There was one focal zone in the proximal endwith relatively thick thrombus associated with inflammation. Microscopicexamination indicated good invasion of the graft. Close to the sutureline the fibroblasts were oriented in a longitudinal manner but theywere randomly oriented further down. There was a lymphocytic,plasmacytic, and fribroblastic response in the region of the suture. Inother areas there was a modest polymorphonuclear reaction within thepre-existing adventitial layer with somewhat less invasion of the graft.There was moderate mural thrombus in other areas. There was extensivecapillary proliferation on the periphery. In general the overallreaction to the graft was good.

Comparable or analogous results to the foregoing may be accomplishedwith various modifications thereof including the following.

A tubular mammalian vessel is used as the starting material, andparticularly preferred is a bovine tubular vessel, including thosehaving a Y or T branch form.

Tubular mammalian vessels prepared and tanned according to the method ofthis invention are particularly suitable for implantation asreplacements for defective vascular, esophagus, bile duct, and uretersegments, but may also be used in sheet form for hernia repair.

The enzyme solution may be commercial ficin material or a purified orconcentrated material containing the proteolytic enzyme and may be usedin a concentration in the range of about 0.25 to 5.0% of active ficin,desirably 0.5 to 2.0%, and preferably 0.5 to 1.5%, the higherconcentration resulting in faster digestion. The digestion temperaturemay be at the range of 30 to 45 C., desirably 34 to 40 C., andpreferably 36 to 38 C.; the higher temperature giving faster digestion.The parts by weight of artery (wet) relative to enzyme solution may bein the range of 1 to 3 per 10 parts of solution, desirably 1 to 2, andpreferably 1.5. The treating solution should be buffered and the pH maybe in the range of 4.0 to 7.0, desirably 4.5 to 6.0, and preferably 5.0to 5.5, the latter giving the faster digestion. The digestion times maybe in the range of 2.0 to 8.0 hours, desirably 2.5 to 6.0, andpreferably 2.5 to 3.0 hours. The concentration, amount, pH, temperatureand time are selected to give the desired increase in collagenous solidscontent.

In collagenous material prepared by enzyme treatment of a mammaliantubular vessel in the above manner, objectionable parenchymatous proteinis removed by enzyme digestion and the collagen fibrils are presentsubstantially in their naturally produced relationship and contain atleast collagenous solids and not over about 20% of other orobjectionable protein based on the weight of solids therein.

The polyacrolein used in the tanning process of this invention may havean average molecular weight within the range of from about 20,000 toabout 500,000, the preferred average molecular weight being within therange of from about 25,000 to about 250,000. If tubular mammalian vesselis tanned with polyacrolein having an average molecular weight belowabout 20,000 or above about 500,000, the tanned tubular mammalian vesselis not tanned s'ufiiciently and upon implantation rapidly dilates andruptures. Polyacrolein may be solubilized by passing gaseous sulfurdioxide with stirring through an aqueous suspension thereof or bystirring a suspension of poly acrolein in an aqueous solution ofsulfurous acid, such as a 7% aqueous sulfurous acid solution, untilsolubilization is complete. Solubilization is complete when the solutiondoes not gel on standing. The concentration of the polyacrolein solutionprepared as above is generally higher than the concentration used intanning a tubular mammalian vessel and may be about 5% (weight/volume).For use in tanning, the solution is diluted to a (weight/volume)concentration of from about 0.2% to about 1.0%, and preferably fromabout 0.4% to about 0.8%. If the concentration of polyacrolein in thetanning solution is less than about 0.1%, tanning is undesirably slowand if the concentration is greater than about 1.0%, no advantage isobtained with respect to degree or speed of tanning. The diluted tanningsolution is adjusted with a base such as sodium hydroxide to a pH offrom about 5 to about 9, and preferably from about 8 to about 9. If thepH is lower than about 5, the walls of the tanned tubular mammalianvessel do not have sufficient rigidity so that rupture occurs onimplantation. If the pH is higher than about 9, the walls are so stiffand rigid there is a tendency to form permanent Wrinkles at flexionpoints which act as foci for thrombus formation and eventual occlusion.The diluted tanning solution may be buttered at the desired pH with anysuitable buffer such as a sodium-potassium phosphate buffer. The use ofa butfered solution is preferred in order that there not be a drop inthe pH of the tanning solution during tanning. Tanning is preferablyaccomplished at room temperature. In order that tanning be stopped, themammalian vessel is removed from the tanning solution and washed withdistilled water until the excess tanning agent is removed. This requiresabout two hours.

About eighteen hours is required for satisfactory tanning of a tubularmammalian vessel by the use of a 0.5% (weight/volume) aqueouspolyacrolein solution, and this is the preferred concentration. Thespeed of tanning is increased with an increase in the concentration ofpolyacrolein in the tanning solution.

Satisfactory tanning is considered to have been accomplished if theshrinkage temperature of the tanned tubular mammalian vessel is withinthe range of from about 83 C. to about 88 C., the preferred shrinkagetemperature being from about 86 C. to about 88 C., and for this purposethe shrinkage temperature is considered to be the temperature at which20% f the potential shrinkage has occurred.

In view of the foregoing disclosures, variations or modificationsthereof will be apparent, and it is intended to include within theinvention all such variations and modifications except as do not comeWithin the scope of the appended claims.

What is claimed is:

1. Collagenous material of mammalian origin tanned with polyacrolein anduseful for implanting in a mamma i 2. Collagenous material of mammalianorigin useful for implanting in a mammal, having its collagen fibrilspresent substantially in their naturally produced relationship andcontaining at least collagenous solids and not over about 20% of otherprotein based on the weight of solids therein and tanned withpolyacrolein having an average molecular weight within the range of fromabout 20,000 to about 500,000 so that it has increased resistance tobreakdown by normal body processes.

3. Collagenous material of mammalian origin useful for implanting in amammal, having its collagen fibrils present substantially in theirnaturally produced relationship and containing at least 80% collagenoussolids and not over about 20% of other protein based on the weight ofsolids therein and tanned with polyacrolein having an average molecularweight within the range of from about 25,000 to about 250,000, andhaving a shrinkage temperature of from about 83 C. to about 88 C.

4. A material of claim 3 in which the shrinkage temperature is fromabout 86 C. to about 88 C.

5. A material of claim 3 in sheet form.

6. A material of claim 3 in tube form.

7. A process for preparing tanned collagenous material of mammalianorigin useful for implanting in a mammal which comprises immersingcollagenous material of mammalian origin which contains at least 80% byweight collagenous solids in an aqueous solution of polyacrolein havinga concentration from about 0.5 to about 10% (weight/volume) and a pHwithin the range of about 5.0 to about 12 for sufficient time to bringthe shrinkage temperature of the tanned collagen to from about 83 C. toabout 88 C.; removing the tanned collagen from the tanning solution andwashing the tanned collagen with distilled water until excess tanningagent is removed therefrom.

8. A process of claim 7 followed by sterilization.

9. A process of claim 8 wherein the sterilization is by means ofpropylene oxide.

References Cited in the file of this patent UNITED STATES PATENTS2,215,453 Buchgraber Sept. 24, 1940 2,583,574 Jones et al. Jan. 29, 19522,640,752 Davis et al. June 2, 1953 2,750,251 Bloch et al. June 12, 19562,886,401 Wells et al. May 12, 1959 2,925,315 Pasternak Feb. 16, 19602,977,182 Adams et al. Mar. 28, 1961 OTHER REFERENCES ].S.L.T.C., vol.24, November 1940, pp. 379-389.

2. COLLAGENOUS MATERIAL OF MAMMALIAN ORIGIN USEFUL FOR IMPLANTING IN AMAMMAL, HAVING ITS COLLAGEN FIBRILS PRESENT SUBSTANTIALLY IN THEIRNATURALLY PRODUCING RELATIONSHIP AND CONTAINING AT LEAST 80% COLLAGENOUSSOLIDS AND NOT OVER ABOUT 20% OF OTHER PROTEIN BASED ON THE WEIGHT OFSOLIDS THEREIN AND TANNED WITH POLYACROLEIN HAVING AN AVERAGE MOLECULARWEIGHT WITHIN THE RANGE OF FROM ABOUT 20,000 TO ABOUT 500,000 SO THAT ITHAS INCREASED RESISTANCE TO BREAKDOWN BY NORMAL BODY PROCESSES.